The Her2+/uPA+ breast cancers are clinically the most aggressive, more so than the Her2+/uPA-, Her2-/uPA+, and Her2-/uPA- subtypes. Proteolytic modulation of Her2/neu/ErbB2 receptor tyrosine kinase signaling via extracellular domain/ectodomain (ECD) shedding by the urokinase-type plasminogen activator (uPA) and a novel network of extracellular membrane serine proteases is hypothesized to play a mechanistic role in the aggressive behaviors of the Her2+/uPA+ breast cancers and in resistance to anti-Her2 therapies using monoclonal antibody or small-molecule tyrosine kinase inhibitor drugs. Targeting Her2 and uPA simultaneously in Her2+/uPA+ breast cancers could therefore be a more effective strategy of treating these cancers;and potentially all Her2+ breast cancers. This hypothesis will be tested in this project. With the following specific aims, we will be creating novel cell-line models of Her2+/uPA+ breast cancer and testing the effectiveness of a novel treatment strategy by targeting Her2 and uPA simultaneously. We will also be investigating novel functional roles of several extracellular membrane serine proteases associated with breast cancer, and many other cancers. Specific Aim 1. To establish Her2+/uPA+ human breast cancer cell lines for evaluation of enhanced aggressiveness. We will establish Her2+/uPA+ human breast cancer cell lines by over-expressing uPA/uPAR in the Her2-amplified SK-BR-3 and BT-474 cell lines or over-expressing Her2 in the uPA+ but Her2-negative (unamplified) MDA-MB-231 cell line;and perform in vitro evaluations of tumor aggressiveness in proliferation, migration, invasion, and anchorage-independent growth. Specific Aim 2. To treat Her2+/uPA+ human breast cancer cell lines with Herceptin, lapatinib, and WX-UK1 for evaluation of effectiveness of targeting Her2 and uPA together. With our engineered Her2+/uPA+ model cell lines, the following clinically relevant questions will be addressed: 1). whether Her2+/uPA+ breast cancers present de novo drug resistance to Herceptin or even lapatinib and whether uPA inhibition with a selective uPA inhibitor WX-UK1 overcomes the resistance;2) whether Her2+/uPA+ breast cancers acquire resistance to anti- Her2 therapies more aggressively and whether uPA inhibition with WX-UK1 delays the acquisition of resistance. Specific Aim 3. To determine the specific actions of matriptase, prostasin, and uPA on Her2 ECD. We will use the FT-293 cell line in co-transfection experiments to determine if uPA is a Her2 ECD sheddase and if plasmin plays a role in Her2 ECD shedding;to purify protease-cleaved Her2 for mapping the cleavage sites;and to determine if the protease-cleaved Her2 is responsive to Herceptin-induced turnover. We will use the SK-BR-3 cell line to determine if the Her2 ECD shedding associated with uPA induction in breast cancer cells also involves or requires matriptase and prostasin by using specific inhibitors. PUBLIC HEALTH RELEVANCE: Resistance to chemotherapy drugs targeting Her2/neu/ErbB2 limits the success of these drugs on select groups of breast cancer patients and could render patients with acquired resistance untreatable. Proteolytic shedding of the Her2 extracellular domain/ectodomain (ECD) by proteases is a mechanism of this resistance. Our preliminary studies have identified new candidate proteases involved in Her2 ECD shedding and the proposed research will reveal if specific protease inhibitors should be considered for combination therapy to reduce or prevent the resistance to the anti-Her2 drugs.